Thermodynamic and structural features of ultrastable

DNA and RNA hairpins

Belen Hernandeza,b, Vladimir Baumrukc, Nicolas Leulliota,b, Catherine Gouyetted,Tam Huynh-Dinhd, Mahmoud Ghomia,b,*

aLaboratoire de Physicochimie Biomoleculaire et Cellulaire (LPBC), UMR CNRS 7033, Universite P. & M. Curie, Case 138,

4 Place de Jussieu, 75252 Paris Cedex 05, FrancebLaboratoire de Physicochimie Biomoleculaire et Cellulaire (LPBC), UMR CNRS 7033, UFR SMBH, Universite Paris 13,

74 rue Marcel Cachin, 93017 Bobigny Cedex, FrancecInstitute of Physics, Charles University, Ke Karlovu 5, 12116 Prague 2, Czech Republic

dUnite de Chimie Organique, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France

Received 3 September 2002; accepted 16 September 2002

Abstract

Short RNA and DNA hairpins have been analysed in aqueous phase by means of UV absorption and vibrational spectroscopy

in the following oligodeoxynucleotide and oligoribonucleotide sequences: 50-d(GC-GAA-GC)-30, 50-r(CGC-GNRA-GCG)-30

(where N ¼ U, A, C, G and R ¼ A, G) and 50-r(GCG-UGAA-CGC)-30. These hairpins contain GAA triloop, GNRA and UGAA

tetraloops stabilised by two or three GC base pairs in their stems. The analysis of UV absorption melting profiles allowed us to

confirm the high (to very high) thermodynamic stability of these hairpins through the estimation of their melting temperature

ðTmÞ: FT-IR spectra revealed the presence of N-type and/or S-type sugar puckers in the hairpins. Raman spectra at the

temperatures below Tm provided information on the conformations of certain nucleosides involved in the hairpins, as well as on

the global conformation (A or B forms) of their stems. Raman spectra recorded as a function of temperature, are consistent with

the hairpin-to-random coil conformational transitions through the breakdown of interbase H-bonds, and the loss of stacking

between the bases. A discussion has been carried out on the agreement between vibrational data and those available from NMR

on a few number of these hairpins.

q 2003 Elsevier Science B.V. All rights reserved.

Keywords: Hairpin; Oligodeoxynucleotide; Oligoribonucleotide

1. Introduction

Hairpins are elementary structural units respon-

sible for nucleic acid (NA) folding. A hairpin consists

of an intramolecular antiparallel double helix (stem)

capped by a certain number of unpaired nucleotides

(loop). In RiboNucleic Acid (RNA), hairpins allow

0022-2860/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved.

PII: S0 02 2 -2 86 0 (0 2) 00 6 27 -0

Journal of Molecular Structure 651–653 (2003) 67–74

www.elsevier.com/locate/molstruc

* Corresponding author. Address: Laboratoire de Physicochimie

Biomoleculaire et Cellulaire (LPBC), UMR CNRS 7033, Universite

P. & M. Curie, Case 138, 4 Place de Jussieu, 75252 Paris Cedex 05,

France. Tel.: þ33-1-442-77555; fax: þ33-1-442-77560.

E-mail addresses: ghomi@lpbc.jussieu.fr (M. Ghomi),

hernandez@lpbc.jussieu.fr (B. Hernandez).

this molecule to fold back on itself and to adopt its

tertiary structures that are necessary for its function in

various biological processes, such as translation and

catalysis. In DeoxyriboNucleic Acid (DNA), hairpins

participate for instance in the formation of cruciform

units. Whatever the type of NA (RNA or DNA) is,

the formation of hairpins leads to an increase of the

structural and thermodynamic stability of the

folded fragments. Hydrogen bonding between

nucleotides and base stacking are basically respon-

sible for the structural and thermodynamic stability

of hairpins.

The size of a loop (number of nucleotides

involved in it) depends on the type of RNA to

which it belongs. Fore instance in transfer RNAs

(tRNAs) the optimal number is seven, whereas in

16S ribosomal RNAs (rRNAs) tetraloops dominate

[1–3]. Furthermore, more than 70% of the tetra-

loops belong to the UNCG and GNRA families

(where N ¼ U, A, C, G and R ¼ A, G). Other

minor tetraloops, such as CUUG and UGAA [3–5],

can also be found in the structure of 16S rRNA. In

this paper, we shall preferentially focus our

attention on RNA tetraloops belonging to GNRA

and UGAA families. For a similar investigation on

UNCG and CUUG tetraloops, the reader is referred

to our previous papers on the subject [6–10]. It

should be recalled that there is a tight connection

between the structure and the function of GNRA

tetraloops. For instance, the GAAA tetraloop

participates in the long range tertiary interactions

in catalytic RNAs [11]; a toxin called ricin

depurinates the second base (A) in the GAGA

tetraloop; the third guanine (G) of this tetraloop

(GAGA) takes part in this RNA/protein interaction

[12]; the GUGA tetraloop is conserved in ribo-

zymes (catalytic RNAs) [13]. Up to now, the use

of high resolution NMR spectroscopy permitted

elucidation of the structural features of the GAAA

[13], GCAA [13], GAGA [13,14], and UGAA [15]

tetraloops formed in short synthetic oligoribonu-

cleotides (ORNs). A schematic representation of

the structural features of GAAA and UGAA

hairpins is given in Fig. 1. The stem of all these

RNA hairpins is formed with a right handed A

form double helix; a mispairing exists between the

first and the last bases of GNRA and UGAA

tetraloops, i.e. GA and UA mispairs, respectively;

the bases are generally stacked but their stacking

depends on the type of the loop fold (Fig. 1).

As far as DNA hairpins are concerned, we limit our

discussion to the case of an ultrastable triloop, i.e.

Fig. 1. Schematic representation of the structural features of (from left to right) the triloop (GAA) and tetraloops (GAAA and UGAA) on the

basis of the previously published NMR data (14–16). Bases are indicated by their first letters (A, U, C, G) and are in anti orientation unless

otherwise indicated. Open pentagon (C20-endo sugar: S-type), filled pentagon (C30-endo sugar: N-type), hatched pentagon (sugar undergoing

C30-endo to C20-endo conformational interconversion), point filled pentagon (sugar that ranges a variety of conformations). Phosphate-

backbone chain is shown by thick lines.

B. Hernandez et al. / Journal of Molecular Structure 651–653 (2003) 67–7468

GAA (Fig. 1), which has been elucidated for the first

time in a pioneering work from Hirao et al. [16] based

on UV absorption and NMR spectroscopy. Although

the stem of this hairpin formed in aqueous phase in a

7-mer oligodeoxynucleotide (ODN) sequence, i.e. 50-

d(GC-GAA-GC)-30, contains only two GC base pairs

(Fig. 1), its Tm value was estimated above 70 8C [16].

Unfortunately, no atomic Cartesian coordinates

derived from NMR structure of the GAA hairpin,

are available to allow its detailed conformational

analysis. However, the comparison of the graphic

representation of GAA triloop (DNA) and GAAA

tetraloop (RNA) allows us to conclude that their basic

structural features are quite similar, i.e. mispairing

between ultimate G and A bases; 50-side stacking of

adenine bases in the both loops. The H-bond networks

in these loops (GAA and GAAA) are different taking

into account their relative sizes (triloop and tetraloop,

respectively). It is, however, difficult to explain the

unusually high value of Tm corresponding to the

above-mentioned short hairpin including a GAA

triloop.

In this report, we mention a representative collec-

tion of our results concerning UGAA tetraloop,

GNRA tetraloops and GAA triloop, obtained by

means of optical spectroscopy (UV absorption,

Raman scattering and FT-IR absorption). These

results allow us to mention and discuss the most

characteristic thermodynamic and structural features

of these stable RNA and DNA hairpins in aqueous

phase.

2. Experimental

2.1. Sample preparation

Ten DNA and RNA oligomers have been syn-

thesized (approximately 5 mg of each sample) at

Institut Pasteur in Paris (France), following the

procedure described previously [10]. These oligomers

correspond to an ODN (7-mer) sequence, 50-d(GC-

GAA-GC)-30, to eight ORN (10-mer) sequences, 50-

r(CGC-GNRA-GCG)-30 (where N ¼ U, A, C, G and

R ¼ A, G) and to an additional ORN (10-mer)

sequence, 50-r(GCG-UGAA-CGC)-30 (Table 1).

Initial lyophilised powder samples contained one

Naþ per phosphate group. They have been dissolved

in a phosphate buffer, pH 6.8, containing 10 mM

monovalent cations (Naþ and Kþ) and 1 mM EDTA,

to obtain aqueous samples used for optical spec-

troscopy. Stock solutions of oligomers with the

following concentrations: Coligomer ¼ 9 mM for

ORNs and 5 mM for ODN, have been first prepared

for recording Raman and IR spectra. For UV

absorption melting profiles, the stock solution was

further diluted in additional phosphate buffer in order

to obtain the following concentrations: Coligomer ¼

100 and 20 mM.

2.2. Spectroscopic measurements

UV absorption melting profiles at 280 nm were

obtained using an UVIKON XL spectrophotometer

with a multi-sample holder, equipped with a Pelletier

heating accessory. Cuvettes with 3 or 4 mm optical

pathlengths, containing oligomer samples were heated

from 10 8C to above 85 8C and then cooled down

for measuring UV absorption profiles with a heating

(or cooling) rate of 0.5 8C/min. Reversible melting

profiles were obtained.

Raman spectra were excited with the 488 or

514.5 nm lines of an argon laser (Stabilite model

2017-04S, Spectra Physics) and collected on a Jobin-

Yvon T64000 spectrograph in a single mono con-

figuration with a 1200 grooves/mm holographic

grating and a holographic notch filter. The spectro-

Table 1

Tm (melting temperature) values of the oligomer sequences forming

stable hairpins in aqueous solutions

Sequences Abbreviation Tm (8C)

Oligoribonucleotides (ORN) tetraloop hairpins

50-r(GCG-UGAA-CGC)-30a UGAAa 46

50-r(CGC-GAGA-GCG)-30 GAGA 51

50-r(CGC-GCGA-GCG)-30 GCGA 56

50-r(CGC-GGGA-GCG)-30 GGGA 58

50-r(CGC-GUGA-GCG)-30 GUGA 58.5

50-r(CGC-GGAA-GCG)-30 GGAA 61

50-r(CGC-GCAA-GCG)-30 GCAA 62

50-r(CGC-GUAA-GCG)-30a GUAAa 63.6

50-r(CGC-GAAA-GCG)-30 GAAA 64

Oligodeoxynucleotide (ODN) triloop hairpin

50-d(GC-GAA-GC)-30a GAAa 73.5

a See Fig. 2 for melting profiles of these hairpins, and see Figs.

3–8 for the vibrational spectra of these hairpins.

B. Hernandez et al. / Journal of Molecular Structure 651–653 (2003) 67–74 69

graph is equipped with a liquid nitrogen cooled CCD

detection system (Spectrum One, Jobin-Yvon) based

on a Tektronix CCD chip of 2000 £ 800 pixels. The

effective spectral slit width was set to ,5 cm21.

Raman spectra were collected in the range 5–90 8C.

Infrared spectra were recorded at room tempera-

ture with a Nicolet Magna 860 FT-IR spectrometer

using a standard source, a CsI beamsplitter and a

DTGS detector. Usually 100 scans were collected

with 4 cm21 spectral resolution and a Happ-Genzel

apodization function. Samples were placed in a

demountable cell (Graseby Specac) consisting of a

pair of ZnSe windows and 12 (or 25) mm Mylar

spacer. All vibrational spectral data were treated

(buffer subtraction, base line correction) using

GRAMS/32 software (Galactic Industries).

3. Results and discussion

For the sake of brevity in this report the oligomer

sequences (ODN and ORNs) are referred to by

recalling their central sequences corresponding to

the triloop or tetraloops that they can form in aqueous

solutions, i.e. GAA (for ODN), GNRA (N ¼ U, A, C,

G and R ¼ A, G) and UGAA (for ORNs), see Table 1

for oligomer sequences and their abbreviations.

3.1. UV absorption melting profiles

UV absorption melting profiles (optical density

versus temperature) obtained for oligomers present all

similar sigmoidal shapes confirming a unimolecular,

progressive and concentration-independent (in the

20–100 mM range) order (hairpin) to disorder (ran-

dom coil) transition (Fig. 2). Tm values (each one

corresponding to the inflection point of a given

melting curve) have been obtained by the second

derivative calculation of the melting profiles (Table

1). These values are located between 46 8C (UGAA

hairpin) and 73.5 8C (GAA hairpin). The GNRA

tetraloop-hairpins possess Tm values ranging from 51

to 64 8C. It should be emphasised that among GNRA

family of tetraloops, two sub-families of tetraloops

can be distinguished, i.e. GNGA hairpins ð51 # Tm #

58:5 8CÞ and GNAA hairpins ð61 # Tm # 64 8CÞ: The

latter result shows that the substitution of a G by an A

base in the third position (R) of a GNRA tetraloop,

leads to an increase of its thermodynamic stability.

3.2. Vibrational spectra

The report of the vibrational spectra of all of the

oligomers studied in this work, is out of the limit of

this paper. Thus, we carry out our discussion on a

representative set of hairpins (belonging to all

families of hairpins), i.e. the UGAA tetraloop

(RNA), GUGA tetraloop (RNA), GUAA tetraloop

(RNA) and GAA triloop (DNA). For the Tm values of

these hairpins, see Table 1.

3.2.1. FT-IR spectra—presence of N-type and S-type

sugar puckers in the hairpins

Figs. 3–5 show the FT-IR spectra of the selected

hairpins recorded in D2O at 20 8C (well below the Tm

of all hairpins) in the 900–725 cm21 spectral region.

These spectra reveal the presence of both S-type (on

the basis of the observed band at ,830 cm21) and N-

type (on the basis of the observed bands at ,812 and

865 cm21) sugars in the GUGA, GUAA hairpins (Fig.

4). This observation can be interpreted by the fact that

the sugar puckers in the middle positions of the

GUGA and GUAA may present a N-type to S-type

Fig. 2. UV absorption profiles (optical density versus temperature)

determined for a selection of four hairpins studied in this work

(UGAA, GUGA, GUAA and GAA). All these curves show a

monophasic, progressive and completely reversible order-to-

disorder transition in the 20–100 mM oligomer concentration

range. For the melting temperatures Tm and abbreviations, see

Table 1.

B. Hernandez et al. / Journal of Molecular Structure 651–653 (2003) 67–7470

(C30-endo to C20-endo) interconversion as confirmed

by the NMR data of GAAA (Fig. 1), GCAA and

GAGA tetraloops (GNRA family) [14]. The UGAA

hairpin does not present the IR marker of S-type sugar

(Fig. 3), despite the fact that corresponding NMR data

predict a variety of sugar puckering for the nucleo-

tides involved in this tetraloop [15] (Fig. 1). On the

basis of the present FT-IR spectra, we can conclude

that the presence of S-type sugars in the UGAA loop

should be ruled out. At last, the GAA triloop presents

both S-type and N-type sugars (Fig. 5). NMR data

[16] have only evidenced the presence of S-type (C20-

endo) sugars in this DNA hairpin. We will continue

our discussion on the presence of N-type sugar in the

GAA hairpin in Section 3.2.3, on the basis of its

Raman spectra.

3.2.2. Raman spectra as a function of temperature—

order to disorder transition of hairpins

Fig. 6 shows the Raman spectra of the selected

oligomers in the 1725–600 cm21 spectral region

recorded at two temperatures located well below and

well above the Tm of the studied hairpins, respectively

(Table 1 and Fig. 2). These spectra are divided into

four contiguous spectral regions (I–IV), each of them

bringing useful information on the order-to-disorder

transition of the studied hairpins.

– In the region I, we can observe the downshift of

the band at ,1710 cm21 (low temperature),

assigned to the base carbonyl stretch, to

,1690 cm21 (high temperature) in RNA oligo-

mers. This effect shows the breakdown of

interbase H-bonds upon increasing temperature.

This effect can be observed to a lesser extent in

the GAA ultarastable triloop-hairpin (only

2 cm21 downshift is observed for the band at

1696 cm21 assignable to the same vibrational

mode). When the temperature is increased,

Raman hypochromism of the G bands at

,1575 and 1485 cm21, is consistent with the

loss of stacking of the G bases involved in the

stem and loop of the hairpins.

– In the region II, we observe intense Raman bands

originating from the nucleoside vibrational

modes. Particularly, the changes observed in

the G band at ,1320 and 1174 cm21, in the

A band at ,1336 cm21, and in the C band at

,1254 cm21 upon increasing temperature,

should be emphasised. All these effects are

consistent with the change in the stacking of

the bases, as well as with the nucleoside

conformations. Other details concerning this

Fig. 3. FT-IR spectra of the UGAA hairpin recorded at room

temperature, D2O buffer (pD 6.8), in the 900–750 cm21 spectral

region. The cytosine ring breathing mode is observed at 783 cm21.

For abbreviations, see Table 1.

Fig. 4. FT-IR spectra of the GUGA and GUAA hairpins (GNRA

family) recorded at room temperature, D2O buffer (pD 6.8), in the

900–750 cm21 spectral region. The cytosine ring breathing mode is

observed at ,780 cm21. For abbreviations, see Table 1.

Fig. 5. FT-IR spectra of the GAA hairpin recorded at room

temperature, D2O buffer (pD 6.8), in the 875–750 cm21 spectral

region. The cytosine ring breathing mode is observed at 784 cm21.

For abbreviations, see Table 1.

B. Hernandez et al. / Journal of Molecular Structure 651–653 (2003) 67–74 71

region will be given in Section 3.2.3.

– In the region III, the Raman bands arising from

the PO22 symmetric stretch band at ,1090 cm21

undergoes a broadening and a downshift upon

increasing temperature. This effect is related to

the order-to-disorder transition of NA chains in

all cases.

– In the region IV, the most interesting result is the

notable decrease of the Raman band observed at

,811 cm21 in RNA hairpins upon increasing

temperature. This band is a well-known Raman

marker for an A form double helix and can be

consequently assigned to the stem of RNA hair-

pins. Raman hypochromism on the breathing

modes of cytosine at ,785 cm21, of adenine at

,727 cm21, and of guanine at,668 cm21 (RNA

hairpins) and at ,680 cm21 (DNA hairpin),

should also be emphasised in this region. This

spectral region will also be detailed in Section

3.2.3.

3.2.3. Raman markers used to determine nucleotide

conformations involved in the hairpins

To describe with more detail the conformational

features of the nucleosides involved in the hairpins,

we present in Figs. 7 and 8 the regions II and IV of

Fig. 6. Only low temperature Raman spectra (con-

cerning hairpins) are displayed.

As mentioned earlier (Section 3.2.1) the DNA

hairpin (GAA) contains both S-type and N-type

conformations. Fig. 7 shows the presence of two

Fig. 6. Raman spectra of a selection of the studied oligomers

(UGAA, GUGA, GUAA, GAA) recorded in H2O buffer (pH 6.8) at

two ultimate temperatures (well below and well above the melting

temperature of each hairpin, respectively). Raman spectra are

excited with 488 nm line (except for the GUAA hairpin, excitation

at 514.5 nm) and displayed in 1725–600 cm21 spectral regions. For

abbreviations, see Table 1.

Fig. 7. Raman spectra of the UGAA, GUGA, GUAA and GAA

hairpins in the 1450–1125 cm21 spectral region. For abbreviations,

see Table 1. This figure corresponds to the region II of Fig. 6 for

only low temperature spectra.

B. Hernandez et al. / Journal of Molecular Structure 651–653 (2003) 67–7472

bands at 1254 and 1267 cm21 assignable to dC

residues with C30-endo/anti and C20-endo/anti con-

formations, respectively. Note that dC residues are

only present in the stem of the GAA hairpin. We

assign the unusual C30-endo/anti conformation (for a

DNA chain in solution) to the 30-terminal dC residue.

It is well known that terminal nucleotides in a hairpin

(or in a double helix), being less stacked compared to

the other bases and in direct contact with water, have a

higher conformational flexibility. However, this

conformational flexibility cannot be confirmed for

the dG residue which is in the 50-terminal of the GAA

hairpin, because Raman spectrum (Fig. 7) presents no

marker band at ,665 cm21 assignable to a dG residue

with C30-endo/anti conformation. We conclude that

all the nucleosides in the GAA tetraloop are in

C20-endo/anti conformation, except the 30-terminal

dC residue which either adopts a C30-endo/anti

conformation, or undergoes a C20-endo/anti to C30-

endo/anti interconversion. The presence of a weak

band at 834 cm21 (B form helix phosphate-backbone

marker), confirms on the other hand that the stem of

the GAA hairpin is a B form mini helix with an

internal structural dynamics due to its 30-terminal dC

residue (mentioned earlier).

In the RNA hairpins only the Raman band at

,1252 cm21 (with no shoulder at ,1265 cm21)

has been observed. On the other hand, the Raman

spectra of RNA hairpins present a band at

668 cm21 (Fig. 8) assignable to C30-endo/anti

rGs. Consequently, on the basis of these two

Raman bands and that observed at ,811 cm21

(A form marker, Fig. 8), we can conclude that

the stem of all these RNA hairpins is a right-

handed A form mini double helix formed by only

three GC base pairs.

NMR data [15] have evidenced the possibility for

the second G of the UGAA tetraloop to adopt a syn

orientation with respect to its adjacent sugar (Fig. 1).

As mentioned earlier (Section 3.2.1) FT-IR spectrum

of this tetraloop (Fig. 3) rules out the presence of S-

type sugars in this hairpin, leading to the conclusion

that all sugars should adopt N-type conformations.

One of the questions raised here is whether the second

rG of the UGAA tetraloop can be precisely in C30-

endo/syn conformation. The response to this question

is negative, because of the low intensity of the rG

Raman marker at 1322 cm21 in this tetraloop (Fig. 7).

It should be emphasised that on the basis of our

previous works on Raman spectra of UNCG tetra-

loops [6–10] containing a C30-endo/syn rG residue at

their ultimate position, as well as on the other works

on Z-form RNA (containing C30-endo/syn rG resi-

dues) [17], the rG residue Raman band at

,1320 cm21 is considerably enhanced when it adopts

a C30-endo/syn conformation. Surprisingly, the

GUGA hairpin (belonging to GNGA family of

tetraloops) manifests an intense band at 1320 cm21.

We can suppose that the second rG residue in the

GUGA tetraloop may adopt a C30-endo/syn confor-

mation, because on the basis of NMR data for GNRA

hairpins (Fig. 1): (i) its sugar can also adopt a C30-

endo conformation (see earlier), (ii) its G base has the

expected flexibility to undergo an anti to syn

interconversion, because it is not involved in an

interbase H-bond network.

Fig. 8. Raman spectra of the UGAA, GUGA, GUAA and GAA

hairpins in the 850–625 cm21 spectral region. For abbreviations,

see Table 1. This figure corresponds to the region IV of Fig. 6 for

only low temperature spectra.

B. Hernandez et al. / Journal of Molecular Structure 651–653 (2003) 67–74 73

4. Conclusion

We have shown above that optical spectroscopy

permits obtention of useful information on the

complex RNA and DNA structures, such as hairpins.

These data can lead to the elucidation of the global

conformation of the stems, N-type and S-type

conformation of sugar puckers, and anti and syn

orientation of the bases with respect to their adjacent

sugars. In many cases, the vibrational data can precise

and complete those obtained by NMR.

Considering the results discussed earlier, our main

conclusion is that vibrational spectroscopy can be

considered as a powerful probe in order to analyse the

overall conformational features of hairpins, probably

before the compilation of time consuming NMR data

with which their detailed 3D structure in aqueous

phase can be accessed.

Acknowledgements

B.H. acknowledges the Spanish Ministry of Edu-

cation, Culture andSport forapost-doctoral fellowship.

V.B. would like to thank the French Ministry of

National Education, Research and Technology for a

PAST-PECO professor fellowship. This work was

partly supported by Czech Ministry of Education,

Youth and Sports (Project No. VS-97113). The authors

thank Nicolas Brunelle for his help in collecting UV

absorption and vibrational data of the GAA hairpin

during his post-graduate training in LPBC.

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